Incorporation Of Phospholipids Research Articles

Abstract PR-06: Dietary fats dictate pancreatic cancer fate via phospholipid saturation

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the US and is anticipated to become the second within the next decade. Epidemiologic studies support a strong link between high fat diet (HFD) consumption and increased PDAC risk, however, the translational relevance of diet research has been limited by inconsistencies in fat source and consumption across human populations and mouse studies. Therefore, whether and how specific dietary fatty acids drive cancer development is poorly understood. To address this gap in knowledge, we performed a diet screen, comprised of 12 isocaloric HFDs differing solely in fat source and representing the diversity of modern human fat consumption, in an oncogenic Kras-driven mouse model (KC: Kras LSL-G12D /+;Pdx1-Cre) that closely mimics the genetic and histologic features of human PDAC progression. Unexpectedly, we discovered that diets rich in oleic acid – a monounsaturated fatty acid (MUFA) typically associated with good health – enhanced pancreatic tumorigenesis. Furthermore, we observed a strong positive correlation between increased tumor burden and diets exhibiting a high ratio of MUFAs to polyunsaturated fatty acids (PUFAs), concordant with high MUFA to PUFA levels in the plasma and in pancreatic phospholipids. Membrane phospholipid incorporation of MUFAs (relative to PUFAs) suppresses lipid peroxidation and cell death via ferroptosis. Consistent with this, pancreata from mice fed a MUFA-rich diet showed decreased expression of genes involved in lipid peroxidation and reduced histologic evidence of lipid peroxidation end products compared to a PUFA-rich diets. Furthermore, primary acinar cells from mice fed high MUFA diets were resistant to cell death upon treatment with ferroptosis inhibitors. Conversely, diets rich in PUFAs increased PUFA incorporation into pancreatic phospholipids, activated transcriptional programs of lipid peroxidation, and intercepted Kras-driven tumor progression in vivo. Collectively, these data support a role for dietary fatty acids in molding the pancreatic phospholipid landscape, altering lipid peroxidation and ferroptosis sensitivity to dictate tumor fate. Citation Format: Christian F Ruiz, Xiangyu Ge, Rylee McDonnell, Daniel McQuaid, Jennifer Kaplan, Guangtao Li, Michael C Rudolph, Fred F Gorelick, John Wysolmerski, Daniel Canals, John D Haley, Matthew Rodeheffer, Mandar D Muzumdar. Dietary fats dictate pancreatic cancer fate via phospholipid saturation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-06.

Metabolic labelling of a subpopulation of small extracellular vesicles using a fluorescent palmitic acid analogue.

Exosomes are among the most puzzling vehicles of intercellular communication, but several crucial aspects of their biogenesis remain elusive, primarily due to the difficulty in purifying vesicles with similar sizes and densities. Here we report an effective methodology for labelling small extracellular vesicles (sEV) using Bodipy FL C16, a fluorescent palmitic acid analogue. In this study, we present compelling evidence that the fluorescent sEV population derived from Bodipy C16-labelled cells represents a discrete subpopulation of small exosomes following an intracellular pathway. Rapid cellular uptake and metabolism of Bodipy C16 resulted in the incorporation of fluorescent phospholipids into intracellular organelles specifically excluding the plasma membrane and ultimately becoming part of the exosomal membrane. Importantly, our fluorescence labelling method facilitated accurate quantification and characterization of exosomes, overcoming the limitations of nonspecific dye incorporation into heterogeneous vesicle populations. The characterization of Bodipy-labelled exosomes reveals their enrichment in tetraspanin markers, particularly CD63 and CD81, and in minor proportion CD9. Moreover, we employed nanoFACS sorting and electron microscopy to confirm the exosomal nature of Bodipy-labelled vesicles. This innovative metabolic labelling approach, based on the fate of a fatty acid, offers new avenues for investigating exosome biogenesis and functional properties in various physiological and pathological contexts.

Phospholipid tail asymmetry allows cellular adaptation to anoxic environments

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Using advanced microscopy, molecular dynamics simulations, and lipidomics by mass spectrometry we demonstrated the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, Schizosaccharomyces japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy, whereas its sister species, the well-known model organism Schizosaccharomyces pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches.

Impact of Sea Warming and 17-α-Ethinylestradiol Exposure on the Lipid Metabolism of Ruditapes philippinarum Clams.

This paper reports on an NMR metabolomics study of lipophilic extracts of Ruditapes philippinarum clams exposed to the hormonal contaminant 17-α-ethinylestradiol (EE2), at 17 °C and 21 °C. The results reveal that exposure at 17 °C triggers a weak response at low EE2 concentrations, suggestive of a slight increase in membrane rigidity, followed by lipid metabolic stability at higher EE2 concentrations. On the other hand, at 21 °C, lipid metabolism begins to respond at 125 ng/L EE2, with antioxidant docosahexaenoic acid (DHA) helping to tackle high-oxidative-stress conditions, in tandem with enhanced storage of triglycerides. Exposure to 625 ng/L EE2 (highest concentration) enhances phosphatidylcholine (PtdCho) and polyunsaturated fatty acid (PUFA) levels, their direct intercorrelation suggesting PUFA incorporation in new membrane phospholipids. This should lead to increased membrane fluidity, probably aided by a decrease in cholesterol. PUFA levels, considered a measure of membrane fluidity, were strongly (and positively) correlated to intracellular glycine levels, thus identifying glycine as the main osmolyte entering the cells under high stress. Membrane fluidity also seems to elicit the loss of taurine. This work contributes to the understanding of the mechanisms of response of R. philippinarum clams to EE2 in tandem with warming while unveiling novel potential markers of stress mitigation, namely high levels of PtdCho, PUFAs (or PtdCho/glycerophosphocholine and PtdCho/acetylcholine ratios) and linoleic acid and low PUFA/glycine ratios.

Engineering phosphatidylinositol-4,5-bisphosphate model membranes enriched in endocytic cargo: A neutron reflectometry, AFM and QCM-D structural study

The combination of in vitro models of biological membranes based on solid-supported lipid bilayers (SLBs) and of surface sensitive techniques, such as neutron reflectometry (NR), atomic force microscopy (AFM) and quartz crystal microbalance with dissipation monitoring (QCM-D), is well suited to provide quantitative information about molecular level interactions and lipid spatial distributions. In this work, cellular plasma membranes have been mimicked by designing complex SLB, containing phosphatidylinositol 4,5-bisphosphate (PtdIns4,5P2) lipids as well as incorporating synthetic lipo-peptides that simulate the cytoplasmic tails of transmembrane proteins. The QCM-D results revealed that the adsorption and fusion kinetics of PtdIns4,5P2 are highly dependent of Mg2+. Additionally, it was shown that increasing concentrations of PtdIns4,5P2 leads to the formation of SLBs with higher homogeneity. The presence of PtdIns4,5P2 clusters was visualized by AFM. NR provided important insights about the structural organization of the various components within the SLB, highlighting that the leaflet symmetry of these SLBs is broken by the presence of CD4-derived cargo peptides. Finally, we foresee our study to be a starting point for more sophisticated in vitro models of biological membranes with the incorporation of inositol phospholipids and synthetic endocytic motifs.

Cancer immune therapy using engineered ‛tail-flipping’ nanoliposomes targeting alternatively activated macrophages

Alternatively-activated, M2-like tumor-associated macrophages (TAM) strongly contribute to tumor growth, invasiveness and metastasis. Technologies to disable the pro-tumorigenic function of these TAMs are of high interest to immunotherapy research. Here we show that by designing engineered nanoliposomes bio-mimicking peroxidated phospholipids that are recognised and internalised by scavenger receptors, TAMs can be targeted. Incorporation of phospholipids possessing a terminal carboxylate group at the sn-2 position into nanoliposome bilayers drives their uptake by M2 macrophages with high specificity. Molecular dynamics simulation of the lipid bilayer predicts flipping of the sn-2 tail towards the aqueous phase, while molecular docking data indicates interaction of the tail with Scavenger Receptor Class B type 1 (SR-B1). In vivo, the engineered nanoliposomes are distributed specifically to M2-like macrophages and, upon delivery of the STAT6 inhibitor (AS1517499), zoledronic acid or muramyl tripeptide, these cells promote reduction of the premetastatic niche and/or tumor growth. Altogether, we demonstrate the efficiency and versatility of our engineered “tail-flipping” nanoliposomes in a pre-clinical model, which paves the way to their development as cancer immunotherapeutics in humans.

High-dose triglyceride DHA supplementation increases plasma and cerebrospinal fluid phospholipid DHA species.

It is not clear whether triglyceride DHA-based formulations (TG-DHA) can sufficiently enrich brain DHA. Animal studies suggest that phosphatidylcholine (PC) and lyso-phosphatidylcholine (LPC) lipids might be preferred brain DHA substrates. The goal of this study is to assess the effect of high-dose TG-DHA supplementation on DHA-containing PC (PC-DHA) and LPC (LPC-DHA) lipid species in plasma and total cerebrospinal fluid (CSF) DHA as a surrogate for brain DHA incorporation. We conducted a randomized clinical trial stratified by DHA treatment (n=14) and placebo (n=14) status in non-demented older participants (CDR ≤ 0.5, age ³ 55 years old). Participants were provided 2,040 mg DHA daily in the form of TG-DHA for six months. Plasma and CSF were obtained at baseline and after supplementation. Participants were also stratified by ApoE ε4 status, an allele known to increase Alzheimer's disease (AD) risk. PC and LPC lipid species from plasma and CSF were isolated and examined by LC/MS-MS. Total fatty acids in CSF were measured via GC/MS-MS. Among all participants, PC-DHA represented 39% of total DHA in plasma, while LPC-DHA represented 1.8% of total DHA. PC-DHA increased by 69% and 19% in plasma and CSF, respectively, following supplementation compared to placebo (p<0.0001 for both). Plasma LPC-DHA increased by 140% in the treatment group compared to placebo (p<0.0001). Increases in plasma PC-DHA and LPC-DHA were both significantly correlated to changes in total DHA in CSF (R=0.8 and 0.57, respectively, p<0.003 for both). A trend toward greater increases in PC-DHA in non-ApoE ε4 carriers compared to carriers were also observed. Our data indicate that high-dose TG-DHA supplementation effectively increases DHA phospholipid incorporation in plasma and CSF. We acknowledge that CSF may not exactly reflect brain lipid uptake but provides useful information on brain DHA bioavailability. Our findings support that high-dose TG DHA supplementation are brain penetrant and can help answer whether enriching brain DHA can slow down cognitive decline in patients at risk of AD.

Suppression of amyloid-β secretion from neurons by cis-9, trans-11-octadecadienoic acid, an isomer of conjugated linoleic acid.

Two common conjugated linoleic acids (LAs), cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA (t10,c12 CLA), exert various biological activities. However, the effect of CLA on the generation of neurotoxic amyloid-β (Aβ) protein remains unclear. We found that c9,t11 CLA significantly suppressed the generation of Aβ in mouse neurons. CLA treatment did not affect the level of β-site APP-cleaving enzyme 1 (BACE1), a component of active γ-secretase complex presenilin 1 amino-terminal fragment, or Aβ protein precursor (APP) in cultured neurons. BACE1 and γ-secretase activities were not directly affected by c9,t11 CLA. Localization of BACE1 and APP in early endosomes increased in neurons treated with c9,t11 CLA; concomitantly, the localization of both proteins was reduced in late endosomes, the predominant site of APP cleavage by BACE1. The level of CLA-containing phosphatidylcholine (CLA-PC) increased dramatically in neurons incubated with CLA. Incorporation of phospholipids containing c9,t11 CLA, but not t10,c12 CLA, into the membrane may affect the localization of some membrane-associated proteins in intracellular membrane compartments. Thus, in neurons treated with c9,t11 CLA, reduced colocalization of APP with BACE1 in late endosomes may decrease APP cleavage by BACE1 and subsequent Aβ generation. Our findings suggest that the accumulation of c9,t11 CLA-PC/LPC in neuronal membranes suppresses the production of neurotoxic Aβ in neurons.

Membrane therapy using DHA suppresses epidermal growth factor receptor signaling by disrupting nanocluster formation

AbstractEpidermal growth factor receptor (EGFR) signaling drives the formation of many types of cancer, including colon cancer. Docosahexaenoic acid (DHA, 22∶6Δ4,7,10,13,16,19), a chemoprotective long-chain n-3 polyunsaturated fatty acid suppresses EGFR signaling. However, the mechanism underlying this phenotype remains unclear. Therefore, we used super-resolution microscopy techniques to investigate the mechanistic link between EGFR function and DHA-induced alterations to plasma membrane nanodomains. Using isogenic in vitro (YAMC and IMCE mouse colonic cell lines) and in vivo (Drosophila, wild type and Fat-1 mice) models, cellular DHA enrichment via therapeutic nanoparticle delivery, endogenous synthesis, or dietary supplementation reduced EGFR-mediated cell proliferation and downstream Ras/ERK signaling. Phospholipid incorporation of DHA reduced membrane rigidity and the size of EGFR nanoclusters. Similarly, pharmacological reduction of plasma membrane phosphatidic acid (PA), phosphatidylinositol-4,5-bisphosphate (PIP2) or cholesterol was associated with a decrease in EGFR nanocluster size. Furthermore, in DHA-treated cells only the addition of cholesterol, unlike PA or PIP2, restored EGFR nanoscale clustering. These findings reveal that DHA reduces EGFR signaling in part by reshaping EGFR proteolipid nanodomains, supporting the feasibility of using membrane therapy, i.e., dietary/drug-related strategies to target plasma membrane organization, to reduce EGFR signaling and cancer risk.

Preparation of nanoliposomes containing HER2/neu (P5+435) peptide and evaluation of their immune responses and anti-tumoral effects as a prophylactic vaccine against breast cancer.

HER2/neu is an immunogenic protein inducing both humoral and cell-mediated immune responses. The antigen-specific cytotoxic T lymphocytes (CTLs) are the main effector immune cells in the anti-tumor immunity. To induce an effective CTL specific response against P5+435 single peptide derived from rat HER2/neu oncogene, we used a liposome delivery vehicle. In vivo enhancement of liposome stability and intracytoplasmic delivery of peptides are the main strategies which elevate the liposome-mediated drug delivery. Liposomes containing high transition temperature phospholipids, such as DSPC, are stable with prolonged in vivo circulation and more accessibility to the immune system. Incorporation of DOPE phospholipid results in the effective delivery of peptide into the cytoplasm via the endocytotic pathway. To this end, the P5+435 peptide was linked to Maleimide-PEG2000-DSPE and coupled on the surface of nanoliposomes containing DSPC: DSPG: Cholesterol with/without DOPE. We observed that mice vaccinated with Lip-DOPE-P5+435 formulation had the highest number of IFN-γ- producing CTLs with the highest cytotoxic activity that consequently led to significantly smallest tumor size and prolonged survival rate in the TUBO mice model. In conclusion, our study indicated that the liposomal form of P5+435 peptide containing DOPE can be regarded as a promising prophylactic anti-cancer vaccine to generate potent antigen-specific immunity.

Дослідження токсичності й антиоксидантної активності ліпосомальних антиоксидантів на моделі Paramecium caudatum

Antioxidant drugs are actively used in medical practice for the treatment of inflammatory processes in various cardiac, ophthalmic, autoimmune, cancer and other diseases. The use of nanobiotechnological methods is a promising area of modern pharmacy, as it allows for creation of drugs of a qualitatively new level. Encapsulation of active pharmaceutical ingredients in liposomal nanoparticles makes it possible to increase the bioavailability and efficacy of natural antioxidants, to create water-soluble injectable forms of hydrophobic compounds. Biotesting is a simple, informative and rapid method to evaluate the toxicity and antioxidant activity of drugs, which can be a valid alternative to the use of laboratory ani­mals at the screening stage. Paramecium caudatum is an unicellular infusoria widely used as test-object due to high sensitivity to environmental changes; big cell size, which makes it possible to monitor changes in morphology and mobility of the cells; easy cultivation. The aim of the study is to evaluate the toxicity and antioxidant activity of liposomal forms of antioxidants: quercetin, curcumin, coenzyme Q10 and cytochrome C by biotesting method using Paramecium caudatum. The toxicity of liposomal forms of quercetin, curcumin, coenzyme Q10 and cytochrome C at doses of 25–100 μg/ml using Paramecium caudatum was studied. Incubation of Paramecium caudatum with liposomal forms of quercetin, curcumin and coenzyme Q10 led to growth of the cell culture, whereas liposomal cytochrome C caused cell lysis within 24 hours. It is established that toxicity of liposomal preparation is influenced by its lipid composition. Incorporation of anionic phospholipid (dipalmitoylphosphatidylglycerol) in lipid membrane significantly reduces the survival of the test culture compared with liposomes containing only phosphatidylcholine. In the model of oxidative stress induced in Paramecium caudatum by hydrogen peroxide, liposomal forms of quercetin, curcumin and coenzyme Q10 demonstrated dose-dependent antioxidant effects, which resulted in tole­rance increasing of the test culture to the toxicant.

Thermosensitive Liposomes Encapsulating Anti-Cancer Agent Lomustine, and Contrast Medium Iohexol, for Thermochemotherapy: Preparation, Characterization, and In Vivo Evaluation.

Thermosensitive liposome-based drug delivery systems (DDS) are powerful tools for site-specific delivery of chemotherapeutics, especially when combined with regional hyperthermia. The objective of this work was to develop a novel thermosensitive liposomal DDS loaded with lomustine, a chemotherapeutic compound, and iohexol, a contrast medium for visualization by CT. Thermosensitive compound liposomes (TSCLs) composed of DPPC were prepared by reverse-phase evaporation and investigated for encapsulation efficiency, temperature-sensitivity, release kinetics, and In Vivo pharmacokinetics. The size and zeta-potential of TSCLs ranged from 250 to 300 nm and -15 to -30 mV, respectively. At 41 °C, TSCLs were shown to release over 90% of iohexol and lomustine within 4 h. The in vitro release profiles of iohexol and lomustine at 41 °C conformed to first-order kinetics and Weibullmodel, respectively. Phase-transition did not occur after incorporation of cholesterol and soybean phospholipids. In Vivo evaluation performed with C6 glioma model rats proved the prolonged half-lives and improved bioavailability by liposomal encapsulation for both compounds under mild local hyperthermia. The TSCLs used in this study may offer a clinically promising mean of increasing efficacy and controlling toxicity.

Lipids and mental disorders: Evidence, uncertainties and perspectives

Brain is an organ with the highest lipid concentration in the body. Cellular membrane lipids can affect both the positioning and the functioning of membrane proteins, thus regulating several cell actions. Changes in the lipid composition of cell membrane can modulate the microenvironment and consequently the function of its proteins, e.g. neurotransmission. Some of the first studies on the subject have shown a negative correlation between serum cholesterol levels and depression, suicidality and behavioral disorders. Several studies -but not all- have found decreased concentrations of ω-3 polyunsaturated fatty acids (ω-3-PUFA) in plasma or erythrocyte membranes of patients with depression, bipolar disorder or after a suicide attempt. In some cases, positive results after their administration have been reported. The effect of ω-3-PUFA in affective disorders is attributed to their action on neurotransmission, neuroplasticity as well as to their anti-inflammatory and anti-oxidative properties. Besides, decreased levels of ω-3-PUFA have been found in erythrocytes or platelets of schizophrenic patients. Some studies have shown in schizophrenics an increased rate of membrane phospholipids breakdown and a decreased ratio of ω-3-PUFA incorporation in phospholipids, possibly because of increased activity of phospholipase A2, an enzyme with crucial role in signaling transduction. Deficient dietetic ingestion of ω-3-PUFA may increase the risk for development of schizophrenia, while a diet rich in ω-3-PUFA may have a preventive role for the disease or improve its course. Although there is no evidence for their action as a monotherapy, they may be useful as an add-on therapy to drug treatment. Some authors suggest that abnormal sphingolipid metabolism, leading to accumulation of ceramides, may be responsible for the development of mood and anxiety disorders, as well as for induction of inflammation or oxidative stress, mechanisms possibly responsible for the physical symptoms of depression. Some drugs seem to combine inhibition of sphingomyelinase (an enzyme catalyzing the production of ceramides) and antidepressant effect. Despite the multitude of related studies, many aspects of the subject remain obscure. Current research focuses on the validity of preventive (especially in the perinatal period) or therapeutic administration of ω-3-PUFA as well as to the pharmacological manipulation of enzymes involved in lipid metabolism (e.g. sphingomyelinase) for the treatment of psychiatric disorders.

Photopolymerized microdomains in both lipid leaflets establish diffusive transport pathways across biomimetic membranes.

Controlled transport within a network of aqueous subcompartments provides a foundation for the construction of biologically-inspired materials. These materials are commonly assembled using the droplet interface bilayer (DIB) technique, adhering droplets together into a network of lipid membranes. DIB structures may be functionalized to generate conductive pathways by enhancing the permeability of pre-selected membranes, a strategy inspired by nature. Traditionally these pathways are generated by dissolving pore-forming toxins (PFTs) in the aqueous phase. A downside of this approach when working with larger DIB networks is that transport is enabled in all membranes bordering the droplets containing the PFT, instead of occurring exclusively between selected droplets. To rectify this limitation, photopolymerizable phospholipids (23:2 DiynePC) are incorporated within the aqueous phase of the DIB platform, forming conductive pathways in the lipid membranes post-exposure to UV-C light. Notably these pathways are only formed in the membrane if both adhered droplets contain the photo-responsive lipids. Patterned DIB networks can then be generated by controlling the lipid composition within select droplets which creates conductive routes one droplet thick. We propose that the incorporation of photo-polymerizable phospholipids within the aqueous phase of DIB networks will improve the resolution of the patterned conductive pathways and reduce diffusive loss within the synthetic biological network.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Formation of a nucleoplasmic reticulum requires de novo assembly of nascent phospholipids and shows preferential incorporation of nascent lamins

Structure of interphase cell nuclei remains dynamic and can undergo various changes of shape and organisation, in health and disease. The double-membraned envelope that separates nuclear genetic material from the rest of the cell frequently includes deep, branching tubular invaginations that form a dynamic nucleoplasmic reticulum (NR). This study addresses mechanisms by which NR can form in interphase nuclei. We present a combination of Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) approach and light microscopy techniques to follow formation of NR by using pulse-chase experiments to examine protein and lipid delivery to nascent NR in cultured cells. Lamina protein incorporation was assessed using precursor accumulation (for lamin A) or a MAPLE3 photoconvertible tag (for lamin B1) and membrane phospholipid incorporation using stable isotope labelling with deuterated precursors followed by high resolution NanoSIMS. In all three cases, nascent molecules were selectively incorporated into newly forming NR tubules; thus strongly suggesting that NR formation is a regulated process involving a focal assembly machine, rather than simple physical perturbation of a pre-existing nuclear envelope.

In vitro and in vivo performance of monoacyl phospholipid-based self-emulsifying drug delivery systems

This study investigates the effect of monoacyl phospholipid incorporation on the in vitro and in vivo performance of self-emulsifying drug delivery systems (SEDDS). Monoacyl phosphatidylcholine (Lipoid S LPC 80 (LPC)) was incorporated into four different fenofibrate (FF)-loaded long-chain SEDDS to investigate the impact of LPC on the emulsion droplet size, extent of digestion, colloidal structure evolution and drug precipitation during in vitro lipolysis simulating human conditions and drug bioavailability in a rat model. The four investigated SEDDS containing long-chain glycerides, polyoxyl 35 castor oil or polyoxyl 8 caprylocaproyl glycerides with or without LPC. In situ synchrotron small/wide-angle X-ray scattering (SAXS/WAXS) was used to simultaneously real-time monitor the kinetics of lamellar phase structure development and FF crystalline precipitation. Adding LPC increased the particle size and polydispersity of the dispersed SEDDS. The two LPC-free SEDDS generated lamellar phase structures (Lα) with d-spacing=4.76nm during digestion. Incorporating LPC into these systems inhibited the formation of lamellar phase structures. The amount of precipitated crystalline FF from the four SEDDS was similar during the first 15min but differed during the last 45min of in vitro digestion. The kinetics of colloidal structure development and FF precipitation was related to the digestion kinetics. The in vivo bioavailability data showed no significant differences between the four SEDDS, which correlates with the in vitro FF precipitation during the first 15min of lipolysis. Thus, the presence of LPC, different emulsion droplet sizes and concentration of lamellar phase structures observed in vitro did not correlate with the FF absorption in rats. The study suggests that later time points of the in vitro lipolysis overestimated FF precipitation in rats because of the high enzyme activity, the lack of gastric and absorption steps, and the low bile salts and phospholipid concentrations of the in vitro model.

Human fetal intestinal epithelial cells metabolize and incorporate branched chain fatty acids in a structure specific manner

BackgroundBranched chain fatty acids (BCFA) are constituents of gastrointestinal (GI) tract in healthy newborn human infants, reduce the incidence of necrotizing enterocolitis (NEC) in a neonatal rat model, and are incorporated into small intestine cellular lipids in vivo. We hypothesize that BCFA are taken up, metabolized and incorporated into human fetal cells in vitro. MethodsHuman H4 cells, a fetal non-transformed primary small intestine cell line, were incubated with albumin-bound non-esterified anteiso-17:0, iso-16:0, iso-18:0 and/or iso-20:0, and FA profiles in lipid fractions were analyzed. ResultsAll BCFA were readily incorporated as major constituents of cellular lipids. Anteiso-17:0 was preferentially taken up, and was most effective among BCFA tested in displacing normal (n-) FA. The iso BCFA were preferred in reverse order of chain length, with iso-20:0 appearing at lowest level. BCFA incorporation in phospholipids (PL) followed the same order of preference, accumulating 42% of FA as BCFA with no overt morphological signs of cell death. Though cholesterol esters (CE) are at low cellular concentration among lipid classes examined, CE had the greatest affinity for BCFA, accumulating 65% of FA as BCFA. BCFA most effectively displaced lower saturated FA. Iso-16:0, iso-18:0 and anteiso-17:0 were both elongated and chain shortened by ±C2. Iso-20:0 was chain shortened to iso-18:0 and iso-16:0 but not elongated. ConclusionsNontransformed human fetal intestinal epithelial cells incorporate high levels of BCFA when they are available and metabolize them in a structure specific manner. These findings imply that specific pathways for handling BCFA are present in the lumen-facing cells of the human fetal GI tract that is exposed to vernix-derived BCFA in late gestation.

Native MS Analysis of Bacteriorhodopsin and an Empty Nanodisc by Orthogonal Acceleration Time-of-Flight, Orbitrap and Ion Cyclotron Resonance.

Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20-0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29-2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.

Lipid Cross-Linking of Nanolipoprotein Particles Substantially Enhances Serum Stability and Cellular Uptake.

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications.